Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/22—Hormones
  • A61K38/31—Somatostatins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/08—Peptides having 5 to 11 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/04—Antineoplastic agents specific for metastasis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P5/00—Drugs for disorders of the endocrine system
  • A61P5/02—Drugs for disorders of the endocrine system of the hypothalamic hormones, e.g. TRH, GnRH, CRH, GRH, somatostatin

Definitions

• Somatostatin a tetradecapeptide discovered by Brazeau et al., has been shown to have potent inhibitory effects on various secretory processes in tissues such as pituitary, pancreas and gastrointestinal tract. SS also acts as a neuromodulator in the central nervous system.
• SSTR1 - SSTR5 G protein coupled receptors
• Somatostatin binds to the five distinct receptor (SSTR) subtypes with relatively high and equal affinity for each subtype.
• SS and various analogues have been shown to inhibit normal and neoplastic cell proliferation in vitro and vivo (Lamberts SW, et al., Endocr Rev 12: 450 - 482) via specific SS receptors (SSTR's) (Patel YC, 1999 Front Neuroendocrinology 20: 157 - 198) and possibly different postreceptor actions (Weckbecker G, et al., Pharmacol Ther 60: 245 - 264; Bell Gl, Reisine T 1993 Trends Neurosci 16: 34 - 38; Patel YC, et al., Biochem Biophys Res Commun 198: 605 - 612; Law SF, et al., Cell Signal 7: 1 - 8).
• SSTR's specific SS receptors
• Binding to the different types of somatostatin receptor subtypes has been associated with the treatment of various conditions and/or diseases.
• the inhibition of growth hormone has been attributed to the somatostatin type-2 receptor ("SSTR2") (Raynor, et al., Molecular Pharmacol. 43:838 (1993); Lloyd, et al., Am. J. Physiol. 268:G102 (1995)) while the inhibition of insulin has been attributed to the somatostatin type-5 receptor ("SSTR5") (Coy, et al. 197:366-371 (1993)).
• Activation of types 2 and 5 have been associated with growth hormone suppression and more particularly GH secreting adenomas (Acromegaly) and TSH secreting adenomas. Activation of type 2 but not type 5 has been associated with treating prolactin secreting adenomas.
• somatostatin receptor subtypes include inhibition of insulin and/or glucagon for treating diabetes mellitus, angiopathy, proliferative retinopathy, dawn phenomenon and nephropathy; inhibition of gastric acid secretion and more particularly peptic ulcers, enterocutaneous and pancreaticocutaneous fistula, irritable bowel syndrome, Dumping syndrome, watery diarrhea syndrome, AIDS related diarrhea, chemotherapy-induced diarrhea, acute or chronic pancreatitis and gastrointestinal hormone secreting tumors; treatment of cancer such as hepatoma; inhibition of angiogenesis; treatment of inflammatory disorders such as arthritis; retinopathy; chronic allograft rejection; angioplasty; preventing graft vessel and gastrointestinal bleeding. It is preferred to have an analog which is selective for the specific somatostatin receptor subtype or subtypes responsible for the desired biological response, thus, reducing interaction with other receptor subtypes which could lead to undesirable side effects.
• MTC medullary thyroid carcinoma
• the present invention relates to the discovery that the human MTC cell line TT, which displays MTC cell characteristics (Zabel M, et al., 1992 Histochemistry 102: 323 - 327, 2 Gagel RF, et al., 1986 Endocrinology 1 18: 1643 - 1651 , Liu JL, et al., 1995 Endocrinology 136: 2389 - 2396) and which stably expresses all the human MTC cell line TT, which displays MTC cell characteristics (Zabel M, et al., 1992 Histochemistry 102: 323 - 327, 2 Gagel RF, et al., 1986 Endocrinology 1 18: 1643 - 1651 , Liu JL, et al., 1995 Endocrinology 136: 2389 - 2396) and which stably expresses all the human MTC cell line TT, which displays MTC cell characteristics (Zabel M, et al., 1992 Histochemistry 102: 323
• SSTR subtypes responds to SSTR2 and SSTR5 activation by subtype selective
• SSTR2 preferential agonists significantly suppress [ H]thy incorporation, i.e., inhibit DNA synthesis, and reduce cell proliferation.
• SSTR5 selective agonists
• SSTR2 and SSTR5 preferential agonists exert differential effects on proliferation of human medullary thyroid TT cell line in vitro, according to their specific SSTR selectivity. Proliferation of the TT cell line can be reduced by SSTR2 selective agonists, but not by SSTR5 agonists, and an SSTR5 agonist can prevent SSTR2 mediated antiproliferative effects.
• SSTR2 selective agonists but not by SSTR5 agonists
• SSTR5 agonist can prevent SSTR2 mediated antiproliferative effects.
• the key inhibitory role of SSTR2 on MTC cell proliferation demonstrates that analogues with enhanced SSTR2 affinity and selectivity versus SSTR5 would be useful as antiproliferative agents in MTC treatment.
• the present invention is based on the discovery that somatostatin agonists selective for SSTR-2 are effective in reducing the rate of proliferation of medullary thyroid carcinoma cells, and that somatostatin agonists selective for SSTR-5 are effective in attenuating this SSTR-2 agonist-induced reduction in rate of proliferation.
• the present invention is directed to a method of modulating the rate of proliferation of MTC cells which comprises contacting MTC cells with one or more SSTR2 agonist and one or more SSTR5 agonist, wherein said SSTR2 agonist serves to reduce the rate of proliferation of the MTC cells and said SSTR5 agonist serves to attenuate the SSTR-2 agonist-induced reduction in proliferation rate.
• the invention is directed to the immediately foregoing method wherein said SSTR-5 agonist is D-Phe-Phe-Trp-D-Trp-Lys-Thr-Phe-Thr- NH 2 or a pharmaceutically acceptable salt thereof.
• the invention is directed to a method of decreasing the rate of proliferation of medullary thyroid carcinoma cells which comprises contacting medullary thyroid carcinoma cells with one or more SSTR2 agonist or a pharmaceutically acceptable salt thereof.
• a method of decreasing the rate of proliferation of medullary thyroid carcinoma cells which comprises contacting medullary thyroid carcinoma cells with one or more SSTR2 agonist or a pharmaceutically acceptable salt thereof.
• SSTR-2 agonist is a SSTR-2 selective agonist.
• the SSTR-2 agonist or pharmaceutically acceptable salt thereof has a Ki value for SSTR-5 that is at least 2 times higher than it has for SSTR-2, more preferably at least 5 times higher than it has for SSTR-2, more preferably still at least 10 times higher than it has for S STR-2.
• the SSTR-2 agonist or pharmaceutically acceptable salt thereof has a Ki value of less than 5 nM, more preferably less than 1 nM.
• the SSTR-2 selective agonist is a compound selected from the list consisting of D-Nal- cyclo[Cys-Tyr-D-Trp-Lys-Nal-Cys]-Thr-NH 2 , cyclo[Tic-Tyr-D-Trp-Lys-Abu- Phe], 4-(2-Hydroxyethyl)- 1 -piperazinylacetyl-D-Phe-cyclo(Cys-Tyr-D-Trp-Lys- Abu-Cys)-Thr-NH 2 , and 4-(2-Hydroxyethyl)- 1 -piperazine-2-ethanesulfonyl-D- Phe-cyclo(Cys-Tyr-D-Trp-Lys-Abu-Cys)-Thr-NH 2 ; or a pharmaceutically acceptable salt thereof, wherein "4-(2-Hydr
• the invention is directed to a method of treating medullary thyroid carcinoma which comprises administering to a patient in need thereof an effective amount of a SSTR2 agonist.
• the SSTR-2 agonist is a SSTR-2 selective agonist.
• the SSTR-2 agonist or pharmaceutically acceptable salt thereof has a Ki value for SSTR-5 that is at least 2 times higher than it has for SSTR-2, more preferably at least 5 times higher than it has for SSTR-2, more preferably still at least 10 times higher than it has for SSTR-2.
• the SSTR-2 agonist or pharmaceutically acceptable salt thereof has a Ki value of less than 5 nM, more preferably less than 1 nM.
• the SSTR-2 selective agonist is a compound selected from the list consisting of D-Nal- cyclo[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH 2 , cyclo[Tic-Tyr-D-Trp-Lys-Abu- Phe], 4-(2-Hydroxyethyl)- 1 -piperazinylacetyl-D-Phe-cyclo(Cys-Tyr-D-Trp-Lys- Abu-Cys)-Thr-NH 2 , and 4-(2-Hydroxyethyl)- 1 -piperazine-2-ethanesulfonyl-D- Phe-cyclo(Cys-Tyr-D-Trp-Lys-Abu-Cys)-Thr-NH 2 ; or a pharmaceutically acceptable salt thereof, wherein "4-(2-Hydroxye
• the invention provides a method of treating medullary thyroid carcinoma patient comprising administering to a patient in need thereof an effective amount of a SSTR2 agonist or a pharmaceutically acceptable salt thereof, wherein said SSTR-2 agonist or pharmaceutically acceptable salt thereof comprises a Tyr(I) residue, wherein the iodine atom of said Tyr(I) residue comprises a radioactive iodine isotope.
• said iodine isotope comprises ' I, I or I.
• medullary thyroid carcinoma cells have formed metastases outside the thyroid.
• said metastases are present in the lymph, the lung, the liver, the brain, or in bone.
• CHO-K1 cells expressing the human SSTR2 were harvested as described in Material and Methods and then the SS analogues (IO 7 - IO '6 M) were added for measurement of intracellular Ca 2+ mobilization, expressed as the ratio between the intracellular calcium concentration measured after the addition of SS analogues and the value observed at basal level.
• the excitation and emission wavelengths were 340 and 510 nm, respectively. The data are represented as mean ⁇ SEM.
• Figure 2 In vitro SSTR5 mediated intracellular calcium mobilization CHO-K1 cells, expressing the human SSTR5, were harvested as described in
• CHO-K1 cells expressing the human SSTR2, were harvested as described in Material and Methods, and then Compound 6 (10 "9 - IO “6 M) and SS (10 nM) were added for measurement of the effect of Compound 6 on SS (10 " M)- stimulated intracellular calcium mobilization, and expressed as the percentage vs. SS alone.
• the excitation and emission wavelengths were 340 and 510 nm, respectively.
• the data are represented as mean ⁇ SEM.
• Figure 4 Somatostatin receptors mRNA expression in TT cells. Extracted RNA (1 ⁇ g/reaction) was treated with deoxyribonuclease and subjected to reverse transcription using Oligo(dT) as primer. Samples incubated without RT enzyme served as control.
• somatostatin receptors e.g., SSTR-1, SSTR-2, SSTR-3, SSTR-4, and SSTR-5.
• a somatostatin agonist may be one or more of an SSTR-1 agonist, SSTR-2 agonist, SSTR-3 agonist, SSTR-4 agonist or a SSTR-5 agonist.
• a somatostatin type-2 receptor agonist i.e., SSTR-2 agonist
• SSTR-2 agonist is a compound which (1) has a high binding affinity (e.g., Ki of less than 100 nM or preferably less than 10 nm or less than 1 nM) for SSTR- 2 (e.g., as defined by the receptor binding assay described below) and (2) decreases the rate of proliferation of medullary thyroid carcinoma cells (e.g., as shown by the biological assay described below).
• a somatostatin type-2 receptor selective agonist is a somatostatin type-2 receptor agonist which has a higher binding affinity (i.e., lower Ki) for SSTR-2 than for SSTR-5.
• a somatostatin type-5 receptor agonist is a somatostatin agonist which ( 1) has a high binding affinity (e.g., Ki of less than 100 nM or preferably less than 10 nm or less than 1 nM) for SSTR-5 (e.g., as defined by the receptor binding assay described below) and (2) attenuates the SSTR-2 agonist-induced decrease in the rate of proliferation of medullary thyroid carcinoma cells (e.g., as shown by the biological assay described below).
• a somatostatin type-5 receptor selective agonist is a somatostatin type-5 receptor agonist which has a higher binding affinity (i.e., lower Ki) for SSTR-5 than for SSTR-2.
• the SSTR-2 agonist is also a SSTR-2 selective agonist.
• the SSTR-2 selective agonist has a Ki value for SSTR-5 that is at least 2 times (e.g., at least 5 times or at least 10 times) higher than it has for the SSTR-2 receptor (e.g., as defined by the receptor binding assay described below).
• SSTR-2 agonists which may be used to practice the present invention include, but are not limited to:
• SSTR-5 agonist which may be used to practice the present invention includes, but is not limited to:
• Patent No.4,369,179 1983 U.S. Patent No.4,360,516 1982
• U.S. Patent No.4,358,439 1982
• U.S. Patent No.4,328,214 1982 U.S. Patent No.4,316,890 1982
• U.S. Patent No.4,310,518 1982 U.S. Patent No.4,291,022 1981
• U.S. Patent No.4,238,481 1980 U.S. Patent No.4,235,886 1980) U.S. Patent No.4,224,199 1980) U.S. Patent No.4,211,693 1980)
• U.S. Patent No.4,146,612 1979 U.S.
• Patent No.4,133,782 1979 U.S. Patent No.5,506,339 1996) U.S. Patent No.4,261,885 1981) U.S. Patent No. 4,728,638 (1988); U.S. Patent No. 4,282,143 (1981); U.S. Patent No. 4,215,039 (1980); U.S. Patent No. 4,209,426 (1980); U.S. Patent No. 4,190,575 (1980);
• each amino acid residue represents the structure of -NH-C(R)H-CO-, in which R is the side chain (e.g., CH 3 for Ala). Lines between amino acid residues represent peptide bonds which join the amino acids. Also, where the amino acid residue is optically active, it is the L-form configuration that is intended unless D-form is expressly designated. For clarity, disulfide bonds (e.g., disulfide bridge) which exist between two free thiols of Cys residues are not shown. Abbreviations of the common amino acids are in accordance with IUPAC-IUB recommendations. Synthesis of somatostatin agonists
• Synthesis of short amino acid sequences is well established in the peptide art.
• synthesis of H-D-Phe-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-NH 2 described above, can be achieved by following the protocol set forth in Example I of European Patent Application 0 395 417 Al .
• the synthesis of somatostatin agonists with a substituted N-terminus can be achieved, for example, by following the protocol set forth in WO 88/02756, European Patent Application No. 0 329 295, and PCT Publication No. WO 94/04752.
• Some of the compounds of the instant invention can have at least one asymmetric center. Additional asymmetric centers may be present on the molecule depending upon the nature of the various substituents on the molecule. Each such asymmetric center will produce two optical isomers and it is intended that all such optical isomers, as separated, pure or partially purified optical isomers, racemic mixtures or diastereomeric mixtures thereof, are included within the scope of the instant invention.
• the compounds of the instant invention generally can be isolated in the form of their pharmaceutically acceptable acid addition salts, such as the salts derived from using inorganic and organic acids.
• acids are hydrochloric, nitric, sulfuric, phosphoric, formic, acetic, trifluoroacetic, propionic, maleic, succinic, D-tartaric, L-tartaric, malonic, methane sulfonic and the like.
• certain compounds containing an acidic function such as a carboxy can be isolated in the form of their inorganic salt in which the counter-ion can be selected from sodium, potassium, lithium, calcium, magnesium and the like, as well as from organic bases.
• the pharmaceutically acceptable salts can be formed by taking about 1 equivalent of a SSTR-2 agonist, e.g., compound 1, and contacting it with about 1 equivalent or more of the appropriate corresponding acid of the salt which is desired. Work-up and isolation of the resulting salt is well-known to those of ordinary skill in the art.
• the compounds of this invention can be administered by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous or subcutaneous injection, or implant), nasal, vaginal, rectal, sublingual or topical routes of administration and can be formulated with pharmaceutically acceptable carriers to provide dosage forms appropriate for each route of administration.
• parenteral e.g., intramuscular, intraperitoneal, intravenous or subcutaneous injection, or implant
• nasal, vaginal, rectal, sublingual or topical routes of administration can be formulated with pharmaceutically acceptable carriers to provide dosage forms appropriate for each route of administration.
• the present invention includes within its scope pharmaceutical compositions comprising, as an active ingredient, at least one SSTR-2 agonist in association with a pharmaceutically acceptable carrier.
• Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.
• the active compound is admixed with at least one inert pharmaceutically acceptable carrier such as sucrose, lactose, or starch.
• Such dosage forms can also comprise, as is normal practice, additional substances other than such inert diluents, e.g., lubricating agents such as magnesium stearate.
• the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings.
• Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, the elixirs containing inert diluents commonly used in the art, such as water. Besides such inert diluents, compositions can also include adjuvants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring and perfuming agents. Preparations according to this invention for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, or emulsions.
• non-aqueous solvents or vehicles examples include propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate.
• Such dosage forms may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. They may be sterilized by, for example, filtration through a bacteria-retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions. They can also be manufactured in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
• compositions for rectal or vaginal administration are preferably suppositories which may contain, in addition to the active substance, excipients such as coca butter or a suppository wax.
• Compositions for nasal or sublingual administration are also prepared with standard excipients well known in the art.
• an effective dosage of active ingredient in the compositions of this invention may be varied; however, it is necessary that the amount of the active ingredient be such that a suitable dosage form is obtained.
• the selected dosage depends upon the desired therapeutic effect, on the route of administration, and on the duration of the treatment, all of which are within the realm of knowledge of one of ordinary skill in the art.
• dosage levels of between 0.0001 to 100 mg/kg of body weight daily are administered to humans and other animals, e.g., mammals.
• a preferred dosage range is 0.01 to 10.0 mg/kg of body weight daily, which can be administered as a single dose or divided into multiple doses.
• MATERIALS AND METHODS RT-PCR analysis was used to demonstrate that all five SSTR subtype mRNA's are expressed in a human MTC cell line, TT.
• the ability of SS analogues with differing affinity and specificity for SSTR2 and 5 subtypes to influence TT cell proliferative activity may be assessed by considering [ H]thy inco ⁇ oration, considered an indirect measure of DNA synthetic activity, and number of viable cells.
• All SSTR2 preferential agonists were able to significantly suppress TT cell number at concentrations ranging from 10 " M to 10 " M.
• Compound 3 and Compound 4 significantly (p ⁇ 0.05) reduced [ 3 H]thy inco ⁇ oration at 10 " M but not at 10 " M and 10 " M, when their maximal inhibitory effect on cell number was apparent.
• Each SSTR2 compound tested showed a trend for decreased efficacy with increasing concentration, however, bell-shaped response curves are common for SS.
• the inhibition of [ 3 H]thy inco ⁇ oration and TT cell number by Compound 1 and Compound 2 at I O "7 M was not associated with any cytotoxic action, as demonstrated by Trypan Blue staining.
• the TT cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).
• the TT cell line consists of aneuploid transformed CT-producing parafollicular cells which are characterised by the presence of a TGC to TGG mutation (Cys to T ⁇ ) at exon 11 codon 634 in the RET protooncogene (Cooley LD, et al., 1995 Cancer Genet Cytogenet 80: 138 - 149), a characteristic that we confirmed in the cell line we worked with.
• TT cells display an impaired expression of the tumor suppressor gene p53 (Velasco JA, et al., 1997 Int J Cancer 73: 449 - 455).
• TT cells express CT and CT receptor (Frendo JL, et al., 1994 FEBS Lett. 342: 214 - 216), carcino-embrionic antigen (CEA), SS, neurotensin, gastrin-releasing peptide (GRP), Leu- and Met-enkephalin, parathyroid hormone releasing peptide (PTH ⁇ ), Chromogranin A, SP-I, Synaptophysin, Neuron- specific enolase (NSE), 1 ,25-dihydroxyvitamin D 3 receptor, Thyrosin hydroxylase, ⁇ -Tubulin, and Cytocheratin (Zabel M, et al., 1995 Histochemical J.
• TT cells secrete a significant amount of CT and respond to changes in ionised calcium levels (Zabel M, et al., 1992 Histochemistry 102: 323 - 327). Thus the TT cell line is suitable for studies on parafollicular function and responses to endocrine and pharmacological stimuli.
• RNA samples were maintained in Ham's Nutrient Mixture F12 with Glutamine (EuroClone Ltd, Torquay, UK), supplemented with 10% fetal bovine serum (FBS, Life Technologies, Milano, Italy), 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 100 ⁇ g/mL amphotericin (EuroClone Ltd, Torquay, UK) at 37°C in a humidified atmosphere of 5% CO 2 and 95% air.
• F12 Ham's Nutrient Mixture
• FBS fetal bovine serum
• streptomycin 100 U/mL
• streptomycin 100 ⁇ g/mL amphotericin
• the TRIZOL protocol is a modification of the guanidinium/phenol extraction. Briefly, the cultured cell media was aspirated and the cells washed with 1 x PBS. The TRIZOL reagent was added and cells lysed at room temperature for 10 min. Chloroform was added to the TRIZOL/cell lysate mixture, and left to stand for 2-3 min, and then centrifuged 12000 x g for 15 min. The aqueous layer was removed from the centrifuged mixture. Isopropanol was added to precipitate the RNA, the pellet collected, washed with 75% ethanol and dried in air.
• RNA was resuspended in diethylpyrocarbonate-treated (DEPC) water and quantified using UV spectrophotometry at 260 nM. To prevent DNA contamination, RNA was treated with ribonuclease-free deoxyribonuclease (Promega, Milano, Italy). RT-PCR Using a first strand complementary DNA (cDNA) synthesis kit
• RNA was reverse transcribed according to the manufacturer's protocol.
• RT mix in PCR tubes was covered with 50 ⁇ l light white mineral oil (Sigma- Aldrich Co ⁇ . Milano, Italy); the RT was carried out in the Minicycler (MJ Research Inc., Watertown, MA, USA) using a program with the following parameters: 10 min at 70°C, 1 min at 4°C, 5 min at 4°C. After supplementing with Superscript II, the reaction was completed at 42°C for 50 min then at 70° for 15 min.
• RNAse H Promega, Milano, Italy
• PCR reactions were carried out using the oligonucleotide primers and the conditions listed in Table 1 , describing the size of expected fragments.
• PCR products were analyzed on a 2% agarose gel and visualized by ethidium bromide (ETB) staining.
• ETB ethidium bromide
• two kinds of negative control were prepared. The first negative control was made by omitting the total RNA in the RT. The second was prepared by replacing the cDNA mix with water in the PCR reaction. The PCR was considered useful only if no band was observed in the negative control lanes on a 2% agarose gel.
• Each PCR product was subjected to restriction enzyme digestion and analysed on 2% agarose gel to further confirm the correct identification of the amplicons.
• SS analogues used in this study and their respective affinities to the different SSTR's are listed in Table 2.
• BSA bovine serum albumin
• Specificity and selectivity of the analogues were determined by Radioligand Binding Assay on CHO-K1 cells stably transfected with each of the SSTR subtypes, as follows.
• Membranes for in vitro receptor binding assays were obtained by homogenizing the CHO-K1 cells expressing the SSTR's subtypes in ice-cold 50 mM Tris-HCl and centrifuging twice at 39000 g (10 min), with an intermediate resuspension in fresh buffer. The final pellets were resuspended in 10 mM Tris- HCl for assay. For the SSTR 1, 3, 4, and 5 assays, aliquots of the membrane preparations were incubated 90 min.
• the final assay volume was 0.3 ml.
• 0.05 nM [ 125 I]MK-678 was employed as the radioligand and the incubation time was 90 min at 25 °C.
• Biological activity of SSTR selective agonists and antagonists was evaluated by the calcium mobilization assay in CHO-K1 cells expressing the human SSTR2 or SSTR5.
• the cells were harvested by incubating in a 0.3% EDT A/phosphate buffered saline solution (25°C), and washed twice by centrifugation. The washed cells were resuspended in Hank's - buffered saline solution (HBSS) for loading of the fluorescent Ca 2+ indicator Fura-2AM.
• HBSS Hank's - buffered saline solution
• Unloaded Fura-2AM was removed by centrifugation twice in HBBS, and the final suspensions were transferred to a spectrofluorometer (Hitachi F-2000) equipped with a magnetic stirring mechanism and a temperature-regulated cuvette holder. After equilibration to 37°C, the SS analogues were added for measurement of intracellular Ca 7+ mobilization. The excitation and emission wavelengths were 340 and 510 nm, respectively. In the SSTR2 expressing cells
• Compound 3 were also highly potent in stimulating Ca mobilization.
• Compound 5 and Compound 1 stimulated significant intracellular Ca 2+ mobilization, whereas Compound 6 displayed slight agonist activity in the range of 300 to 1000 nM.
• Compound 6 inhibited SS-induced intracellular Ca + mobilization in SSTR2 expressing cells in a dose dependent manner with complete suppression of
• TT cells were plated in 24-multiwell plates (10 5 cells/well) and incubated for 48 hours in a medium supplemented with 10% FBS in the presence of [ H]thy (1.5 ⁇ Ci/mL; 87 Ci/mmol) with or without each SS analogue at concentrations ranging from 10 " to IO “9 M. Treatments were renewed by adding fresh analogues to the wells after the first 24 h of incubation, without removing the medium.
• TCA-precipitated material was solubilized in 500 ⁇ L 0.2 mol/L sodium hydroxide and 0.1% SDS. Cell- associated radioactivity was then counted in a scintillation spectrometer. Results (counts per min per well) were obtained by determining the mean value of at least six experiments in quadruplicate. The viability of TT cells in control and treated cultures was evaluated by Trypan blue staining both after 24 and 48 hours, and the number of viable cells was always 85 -95%.
• the effects of SSTR selective agonists and antagonists on TT cell proliferation were assessed by the CELLTITER 96 Aqueous Non-Radioactive Cell Proliferation Assay (Promega, Milano, Italy), a colorimetric method for determining the number of viable cells in proliferation assays.
• the assay contains solutions of a tetrazolium compound (Owen's reagent; MTS) and an electron coupling reagent (phenazine methosulphate; PMS).
• MTS tetrazolium compound
• PMS phenazine methosulphate
• the absorbance of the formazan at 490 nm can be measured directly from 96 well assay plates (Zatelli MC, et al., 2000 J Clin Endocrinol Metab 85: 847 - 852; Cory AH, et al., 1991 Cancer Conmiun 3: 207 - 212).
• the conversion of MTS into the aqueous soluble formazan is accomplished by dehydrogenase enzymes found in metabolically active cells.
• the quantity of formazan product as measured by the amount of 490 nm absorbance is directly proportional to the number of living cells in culture.
• TT cells were plated in 96-multiwell plates (2 x 10 cells/well) and incubated for 48 hours in a medium supplemented with 10% FBS in the presence or absence of each SS analogue at concentrations ranging from IO "6 to 10 " M. Treatments were renewed by adding fresh analogues to the wells after the first 24 hours of incubation. At the end of the incubation period, 20 ⁇ l of a combined MTS/PMS solution were added to each well with a repeating pipette, and the plates were incubated for an additional 4 hours at 37°C in a humidified 5% CO 2 atmosphere.
• TT cells express SSTR's that could mediate a potential response to selective compounds for individual SSTR subtypes.
• RNA was isolated from cultured TT cells and performed RT-PCR reactions in the conditions described in Material and Methods. Integrity of cDNA was assured by the presence of the GAPDH signal. The absence of genomic DNA contamination in the cDNA samples was assessed by the lack of any amplification in a PCR reaction using non-reverse transcribed samples. Positive amplification of SSTRl, 2, 3, 4, and 5 was found in the examined cell line (Fig.4), demonstrating that these receptors are expressed in human MTC cell-line TT. The demonstration that the TT cell line stably expresses SSTR subtypes made this cellular model system suitable for evaluating the action of receptor-selective SS analogues.
• TT cell [ HJthy inco ⁇ oration and proliferation were examined testing each of Compound 2 and Compound 5 at 10 " M in combination with increasing doses (from IO "9 M to 10 " M) of the other compound.
• the results are summarized in Figure 8.
• Table 2 Human somatostatin receptor subtype specificity (ICsn. nM)
• Subtype affinity was determined by radioligand membrane receptor binding assays in Chinese hamster ovary cells expressing human SSR2 gene or SSR5 cDNA

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• Endocrinology (AREA)
• Zoology (AREA)
• Oncology (AREA)
• Diabetes (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Peptides Or Proteins (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Steroid Compounds (AREA)
• Medicines Containing Material From Animals Or Micro-Organisms (AREA)
• Investigating Or Analysing Biological Materials (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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• 2002
  • 2002-03-06 CA CA2743731A patent/CA2743731C/en not_active Expired - Fee Related
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  • 2002-03-06 RU RU2007111864/04A patent/RU2352578C2/ru active
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  • 2002-03-06 WO PCT/US2002/006729 patent/WO2002070555A2/en active IP Right Grant
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  • 2002-03-06 RU RU2003129515/14A patent/RU2275934C2/ru active
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• 2003
  • 2003-09-02 NO NO20033881A patent/NO20033881L/no not_active Application Discontinuation
• 2005
  • 2005-10-20 RU RU2005132456/14A patent/RU2317824C2/ru active
• 2007
  • 2007-01-15 JP JP2007005725A patent/JP4828438B2/ja not_active Expired - Fee Related
  • 2007-05-02 US US11/799,594 patent/US9220760B2/en not_active Expired - Fee Related
  • 2007-06-05 US US11/810,268 patent/US20110183416A1/en not_active Abandoned

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